Methods and compositions for isolation and growth of kidney tubule stem cells, in vitro kidney tubulogenesis and ex vivo construction of renal tubules

Abstract

Methods, including culture media conditions, which provide for isolation and purification of renal tubule stem cells and for in vitro kidney tubulogenesis are disclosed. The methods rely on culturing adult kidney cells in a culture media treated with combinations of transforming growth factor- beta 1, epidermal growth factor, and all-trans retinoic acid.

What is claimed as new and desired to be secured by Letters Patent of the United States is: 
     
       1. An ex vivo renal tubule tissue system prepared by a process comprising culturing kidney cells in a culture medium comprising all-trans retinoic acid, transforming growth factor-β 1  and either epidermal growth factor or transforming growth factor-α in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       2. The renal tubule tissue system of claim 1, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, selenium, fibroblastic growth factor, hepatocyte growth factor and. 
     
     
       3. The renal tubule tissue system of claim 1, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       4. The renal tubule tissue system of claim 3, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor and hepatocvte growth factor, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       5. The renal tubule tissue system of claim 1, wherein said culture medium contains an adhesion molecule. 
     
     
       6. The renal tubule tissue system of claim 1, wherein said culture medium comprises a soluble factor selected from the group consisting of insulin-like growth factor I, Wnt-1 and Wnt-4. 
     
     
       7. The renal tubule tissue of claim 1, wherein said culture medium contains an adhesion molecule. 
     
     
       8. The method of claim 7, wherein said adhesion molecule presents multiple copies of the Arg-Gly-Asp cell attachment sequence from human fibronectin where which has an Ile-Lys-Val-Ala-Val epitope from the laminin alpha chain. 
     
     
       9. A method for growing kidney tubule stem cells ex vivo, comprising culturing kidney cells in the presence of all-trans retinoic acid, transforming growth factor-β 1 , and either epidermal growth factor or transforming growth factor-α, in amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       10. The method of claim 9, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor. 
     
     
       11. The method of claim 9, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       12. The method of claim 9, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       13. The method of claim 9, wherein said culture medium contains an adhesion molecule. 
     
     
       14. The method of claim 13, wherein said adhesion molecule presents multiple copies of the Arg-Gly-Asp cell attachment sequence from human fibronectin where which has an Ile-Lys-Val-Ala-Val epitope from the laminin alpha chain. 
     
     
       15. A method of expressing a therapeutically useful polypeptide in a renal tubule system comprising transforming a renal cell;   culturing the transformed renal cell ex vivo in the presence of all-trans retinoic acid and either epidermal growth factor and transforming growth factor-α in an amount for achieving tubulogenesis; and   culturing the transformed renal tubule cells for a time sufficient to express a therapeutically useful polypeptide.   
     
     
       16. The method of claim 15, wherein the transformed renal tubule cells comprise kidney tubule stem cells stably genetically transformed with DNA encoding the therapeutically useful polypeptide. 
     
     
       17. The method of claim 16, wherein the transformed renal tubule cells further comprise kidney tubule progenitor cells stably genetically transformed with DNA encoding the therapeutically useful polypeptide. 
     
     
       18. The method of claim 15, wherein the transformed renal tubule cells comprise kidney tubule progenitor cells stably genetically transformed with DNA encoding the therapeutically useful polypeptide. 
     
     
       19. The method of claim 15, wherein the culturing step is performed in the presence of at least one culture medium comprising a soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor. 
     
     
       20. The method of claim 15, wherein the culturing step is performed in the presence of a culture medium comprising at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       21. The method of claim 15, wherein the culturing step is performed in the presence of a culture medium comprising at least one soluble factor and at least one insoluble factor, wherein said soluble factor is selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor, and wherein said insoluble factor is selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       22. A method of expressing a therapeutically useful polypeptide in an ex vivo renal tubule system, comprising: transforming an ex vivo renal tubule system prepared by a process comprising culturing kidney cells in a culture medium comprising all-trans retinoic acid, transforming growth factor-β1 and either epidermal growth factor or transforming growth factor-α in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border; and   culturing the transformed ex vivo renal tubule system for a time sufficient to express a therapeutically useful polypeptide.   
     
     
       23. The method of claim 22, wherein the culture medium further comprises at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor. 
     
     
       24. The method of claim 22, wherein the culture medium further comprises at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       25. The method of claim 22, wherein the culture medium further comprises at least one soluble factor and at least one insoluble factor, wherein said soluble factor is selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, selenium, fibroblastic growth factor, and hepatocyte growth factor, and wherein said insoluble factor is selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       26. An ex vivo renal tubule tissue system prepared by a process comprising culturing kidney cells in a culture medium comprising all-trans retinoic acid and either epidermal growth factor or transforming growth factor-α in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       27. The renal tubule tissue system of claim 26, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       28. The renal tubule tissue system of claim 26, wherein said culture medium comprises at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, and selenium. 
     
     
       29. The renal tubule tissue system of claim 26, wherein said culture medium comprises at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone triiodothyronine, and selenium, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       30. A method for growing kidney tubule stem cells ex vivo, comprising culturing kidney cells in the presence of all-trans retinoic acid and either epidermal growth factor or transforming growth factor-α, in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       31. The method of claim 30, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       32. The method of claim 30, wherein said culture medium comprises at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, and selenium. 
     
     
       33. The method of claim 30, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, and selenium, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       34. A method for effecting tubulogenesis in a renal cell culture ex vivo, comprising culturing kidney cells in the presence of all-trans retinoic acid and either epidermal growth factor or transforming growth factor-α in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       35. The method of claim 34, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       36. The method of claim 34, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, and selenium. 
     
     
       37. The method of claim 34, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, and selenium, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       38. A method for constructing and maintaining an ex vivo renal tubule tissue system, comprising culturing kidney cells in the presence of all-trans retinoic acid and either epidermal growth factor or transforming growth factor-α in an amount effective for achieving tubulogenesis, wherein tubulogenesis is a phenotypic transformation of said cells characterized by condensed aggregates of tubule cells forming about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype with extensive microvilli formation and tight junctional complexes along the lumenal border. 
     
     
       39. The method of claim 38, wherein said culture medium contains at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin. 
     
     
       40. The method of claim 38, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triodothyronine, and selenium. 
     
     
       41. The method of claim 38, wherein said culture medium contains at least one soluble factor selected from the group consisting of fetal calf serum, prostaglandins, hydrocortisone, triiodothyronine, and selenium, and at least one insoluble factor selected from the group consisting of Type I collagen, Type IV collagen, laminin, proteoglycans, and fibronectin.